During the late phase of HIV-1 infection, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on specific interactions between its matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a signaling lipid localized on the inner leaflet of the PM. Concurrent or subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles, a process that is dependent on Gag assembly. Several lines of evidence suggest that incorporation of Env is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of gp41 (gp41CT), a mechanism that remains to be elucidated. For over a decade, we have pioneered approaches to investigate at the molecular level how retroviral (HIV-1, HIV-2, MLV, and ASV) MA domains of Gag interact with lipids (e.g., PI(4,5)P2) and membranes, a key requirement for understanding Gag assembly and ultimately Env incorporation. A major barrier to characterizing gp41CT?MA interaction has been the unavailability of a recombinant gp41CT protein and the inability to reconstitute the proper conditions for the interaction to occur. During this funding period we have recorded a breakthrough by determining the three- dimensional structure of HIV-1 gp41CT and characterized its interaction with membrane. This achievement, an elusive task for over two decades, filled a major gap by providing the structure of the last segment of HIV-1 proteome. In this renewal, we set our sights on elucidating the molecular mechanism by which Gag mediates the recruitment of HIV-1 Env into assembly sites. We will test the hypothesis that trimerization of the MA domain plays an important role in gp41CT interaction and therefore Env recruitment into particles. We will employ a battery of structural biology, biophysical, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to membrane and how it interacts with gp41CT. Our specific aims are: (i) Engineer HIV-1 MA trimer and hexamer and characterize their binding to membranes, (ii) characterize the interactions between HIV-1 MA and gp41CT, and (iii) determine the structure of MA?gp41(TM-CT)?membrane complex by single-particle cryo-EM. This proposal rests on a solid premise of: (1) 25 years of functional data that is waiting for a molecular interpretation to provide sharper insights into Gag assembly on the PM and mechanisms of Env incorporation, (2) highly technical skill sets for working with proteins, membranes, structural and biophysical tools, (3) strong preliminary data, and (4) an outstanding collaborative team with 20-30 years of experience in cryo-EM, x-ray crystallography, and mass spectrometry. Therefore, the proposed structural studies will provide a detailed understanding of HIV-1 Gag assembly on the PM and Gag?mediated Env incorporation. We hope that the outcome of this proposal will help in the development of new antiviral therapeutic agents that inhibit assembly, Env incorporation and ultimately virus production.